S247: EMERGENCY GRANULOPOIESIS INDUCES A LYMPHOID TO MYELOID BIAS SWITCH IN A SUBSET OF HEMATOPOIETIC STEM CELLS
Authors: K. Vanickova, P. Danek, M. Milosevic, M. K. Adamcova, N. Rai, F. Matteini, M. C. Florian, J. D’Armiento, J. Rohlena, K. Rohlenova, M. Alberich-Jorda
Journal: HemaSphere (2022)

Background: Granulocytes represent the first line of defense against bacteria and fungi, thus their production needs to be adapted to specific demands. While daily production of granulocytes is sustained by steady-state granulopoiesis, upon infection this program switches to emergency granulopoiesis (EG), to ensure enhanced and accelerated granulocytic production. Recently, it was shown that unperturbed hematopoiesis is sustained by multipotent progenitors, however, during stress conditions, hematopoietic stem cells (HSCs) become active and responsible for coping with stress situations. Yet, whether and how HSCs play a role during EG is unknown. Aims: The aim is to understand the role of HSCs at early stages of acute infection, and to determine the mechanisms that drive emergency granulopoiesis at the stem cell level. Methods: The methods used in the project include single cell RNA sequencing, qRT-PCR, ELISA, whole-mount bone marrow microscopy, flow cytometry sorting and analysis, stem cell in vitro cultures, Wnt10b knockout mouse model and murine LPS administration. Results: To understand the contribution of HSCs to the EG response, we performed single cell RNA sequencing analysis of sorted murine HSCs (Lin-c-Kit+Sca-1+CD48-CD150+) 4 hours after lipopolysaccharide (LPS) stimulation, which mimics a bacterial infection and activates EG in vivo. Strikingly, we observed radical changes in the HSC cluster composition between PBS control and LPS treated mice. Interestingly, these changing population dynamics were marked by alterations in HSC lineage bias, demonstrating that under EG there is an expansion of the myeloid-bias HSCs at expenses of the lymphoid-bias HSCs. Remarkably, we identified Procr (CD201) as a specific cell surface marker for this lymphoid to myeloid transition, allowing us to distinguish, sort and track the switch from CD201+ lymphoid-bias HSCs (present in PBS control) towards CD201- myeloid-bias HSCs (present upon LPS stimulation). Further, the abundant CD201- myeloid-bias HSC clusters were characterized by an inflammatory gene signature. Particularly interesting, was the elevated expression of Wnt10b. We verified that Wnt10b is upregulated in murine primary bone marrow cells upon LPS stimulation in vitro using qRT-PCR. Further, we demonstrated that Wnt10b is released in the bone marrow following LPS treatment in vivo using ELISA and whole-mount microscopy. We showed that Wnt10b activates the canonical Wnt/β-catenin signaling pathway and promotes myeloid differentiation of Lin-c-Kit+Sca-1+ cells in vitro. In addition, our preliminary data suggests that the EG response is impaired in Wnt10b KO mice following LPS stimulation in vivo. Summary/Conclusion: In conclusion, we observed that HSCs actively take part in early stages of EG by activating an inflammatory signature associated with the switch from lymphoid to myeloid bias, therefore ensuring sufficient production of myeloid cells to fight the infection. Acknowledgements: This work was partially supported by a GACR grant 22-18300S and institutional funding from the IMG CAS (RVO 68378050).